Construction of rabbit Hypoxanthine guanine phosphoribosyl transferase gene-targeting vector by Red homologous recombination System
利用Red同源重组系统构建兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因打靶载体

It has been reported that a gene can be knocked out by homologous recombination technology in EL350 genetically engineered bacteria strain. However, the study about the mutation and genetic targeting of Hypoxanthine Guanine Phosphoribosyl Transferase (HPRT) gene by this system has not been reported. In this paper, rabbit full length HPRT gene BAC clone LBNL1-304M19 is used as the template. A 47kb rabbit HPRT gene fragment, which does not have promoter and exon1, is cloned into pBACLinkSp plasmid to form pBACLinkSp-rHPRT recombinant plasmid via Gap-Repair by Red recombination system. Then, different homologous arms are designed to delete different coding region of HPRT gene on the basis of the pBACLinkSp-rHPRT plasmid. Three different HPRT gene targeting vectors have been constructed. Meanwhile, the efficiency of deleting different sizes of DNA fragment by homologous recombination technology has also been studied. These three different HPRT gene targeting vectors form the basis for exploring the gene targeting in rabbit fibroblast cells and embryonic stem cells, and making rabbit HPRT gene knockout models in the future.